The Mechanism Of P53/mir-22/tet2 Pathway In Acute Myeloid Leukemia

Objective: This study explored the mechanism of tet2 mediated by wild-type p53 gene as a transcription factor regulating mir-22 in acute myeloid leukemia.To explore the clinical significance of the interaction regulation function and molecular mechanism among the three for acute myeloid leukemia.Methods:(1)To design mir-22 knockdown and overexpression interference vectors to transfect K562 cells,and use qRT-PCR to detect the expression level of mir-22 to verify the success of cell transfection.(2)After verifying the success of cell transfection,48 hours after cell transfection,Western Blot method was used to detect the expression levels of p53 and tet2 proteins under mir-22 knockdown and overexpression,and qRT-PCR was used to detect the expression levels of p53 and tet2 genes;(3)Knock down the tet2 interference vector and transfect K562 cells.Observe the green fluorescence and the qRT-PCR method to detect the expression level of tet2 using a fluorescence microscope to verify the success of the transfection.Using qRT-PCR method to detect the expression levels of p53 and mir-22 before and after knocking down tet2.(4)CCK-8(Cell counting Kit-8)method was used to detect the knockdown and overexpression of mir-22 and tet2 after knockdown K562 cell activity;(5)Flow cytometry was used to detect the apoptosis of K562 after knocking down and overexpressing mir-22 and after knocking down tet2 and its effect on cell cycle regulation;(6)Using dual luciferase gene experiment to verify p53 as Transcription factors target the mir-22 promoter and mir-22 binds tet2.Results:(1)The experimental data showed that the expression rate of mir-22 gene in K562 cells decreased by 80% after knocking down mir-22(P <0.01),and the expression level of mir-22 gene increased 9311 times after overexpression(P <0.001).It shows that the cell knockdown and overexpression are successful;(2)QRT-PCR and Western Blot test results showed that: mir-22 knockdown can promote the expression of tet2 protein and gene and inhibit the expression of p53 protein and gene in K562 cells;Expression inhibits the expression of tet2 protein and gene,and promotes the expression of p53 protein and gene in K562 cells.After tet2 knockdown,the results showed that the expression of p53 gene and protein increased,and the expression of mir-22 gene decreased;(3)CCK8 test results showed that overexpression of mir-22 in K562 cells resulted in a significant decrease in cell proliferation rate,while mir-22 knockdown detected an increase in cell proliferation rate compared with the control group at 48 h,which was statistically significant;(4)Flow cytometry detects the number of cells arrested in G0 / G1 phase after knocking down mir-22,and promotes the increase of cells in S phase;after overexpressing mir-22 and knocking down the tet2 gene,the number of cells stalled in G0 / G1 phase Increased,inhibited cell tumor cells from dividing and promoted apoptosis,which was statistically significant;(5)Dual luciferase test showed that the dual luciferase activity of p53 + mir-22 promotor-WT group decreased(P <0.01),Suggesting that p53 inhibits the expression of mir-22.The dual luciferase activity of the mir-22 mimics + tet2 3'UTR group decreased(P <0.01).Conclution:(1)Overexpression and knockdown of mir-22 in acute myeloid leukemia have an effect on cell proliferation,apoptosis and cell cycle,and mir-22 has an inhibitory effect on the occurrence and development of AML;(2)p53 targeting Act on mir-22 and inhibit its expression;mir-22 targets tet2 through tet2 3?UTR end;(3)After overexpression of mir-22,it can inhibit tet2 gene expression and protein synthesis,thereby promoting increased expression of p53 gene and protein It leads to a decrease in the expression level of mir-22;(4)The existence of p53 / mir-22 / tet2 pathway in acute myeloid leukemia,which may have an important role in its pathogenesis.If we further in-depth research in acute Myeloid leukemia maybe will seek new therapeutic targets,develop new drugs to improve the overall survival rate and quality of life of patients,reduce the economic burden of patients,and create economic benefits for society.